Molecular testing for gastrointestinal pathogens in intestinal tissue of infants with necrotizing enterocolitis or spontaneous intestinal perforation.

OBJECTIVE: The objective of this study was to determine the frequency of common gastrointestinal bacterial, parasitic, and viral pathogen detection in necrotizing enterocolitis (NEC) or spontaneous intestinal perforation (SIP) -associated intestinal tissue. STUDY DESIGN: Retrospective cohort study examined formalin fixed, paraffin embedded (FFPE) surgical or autopsy intestinal tissue from NEC or SIP specimens. DNA and RNA were extracted and analyzed by multiplex PCR panel (GIFA Biofire). DNA or RNA from stool samples containing each pathogen were extracted for positive controls. RESULTS: The total number of intestinal tissue samples were 193 from 310 infants (156 NEC, 37 SIP). Six (3%) infants with stage III NEC tested positive for a target pathogen; 2, C. difficile; 3, Enteroaggregtive E. coli; and 1, Giardia. No gastrointestinal viral pathogens were detected. CONCLUSION: Molecular testing yielded few GI pathogens suggesting that these organisms are likely not major causes or facilitators of NEC or SIP.

Assessment of Beta-2 Microglobulin Gene Edited Airway Epithelial Stem Cells as a treatment for Sulfur Mustard Inhalation.

Respiratory system damage is the primary cause of mortality in individuals who are exposed to vesicating agents including sulfur mustard (SM). Despite these devastating health complications, there are no fielded therapeutics that are specific for such injuries. Previous studies reported that SM inhalation depleted the tracheobronchial airway epithelial stem cell (TSC) pool and supported the hypothesis, TSC replacement will restore airway epithelial integrity and improve health outcomes for SM-exposed individuals. TSC express Major Histocompatibility Complex (MHC-I) transplantation antigens which increases the chance that allogeneic TSC will be rejected by the patient's immune system. However, previous studies reported that Beta-2 microglobulin (B2M) knockout cells lacked cell surface MHC-I and suggested that B2M knockout TSC would be tolerated as an allogeneic graft. This study used a Cas9 ribonucleoprotein (RNP) to generate B2M-knockout TSC, which are termed Universal Donor Stem Cells (UDSC). Whole genome sequencing identified few off-target modifications and demonstrated the specificity of the RNP approach. Functional assays demonstrated that UDSC retained their ability to self-renew and undergo multilineage differentiation. A preclinical model of SM inhalation was used to test UDSC efficacy and identify any treatment-associated adverse events. Adult male Sprague-Dawley rats were administered an inhaled dose of 0.8 mg/kg SM vapor which is the inhaled LD50 on day 28 post-challenge. On recovery day 2, vehicle or allogeneic Fisher rat UDSC were delivered intravenously (n = 30/group). Clinical parameters were recorded daily, and planned euthanasia occurred on post-challenge days 7, 14, and 28. The vehicle and UDSC treatment groups exhibited similar outcomes including survival and a lack of adverse events. These studies establish a baseline which can be used to further develop UDSC as a treatment for SM-induced airway disease.

Factors Associated with Neurodevelopmental Impairment in Bronchopulmonary Dysplasia.

OBJECTIVE: To identify factors associated with neurodevelopmental impairment (NDI) in patients with bronchopulmonary dysplasia (BPD). STUDY DESIGN: We identified 151 patients with moderate to severe BPD from 2010 to 2014 with complete Bayley Scales of Infant Development (BSID) scores at 24 months corrected age. We defined NDI as any diagnosis of cerebral palsy or ≥1 BSID composite scores of <80. RESULTS: The mean corrected age at BSID was 23 ± 1 months; 18% had a cognitive score of <80, 37% had a communication score of <80, and 26% had a motor score of <80. Cerebral palsy was diagnosed in 22 patients (15%); 84 (56%) patients did not have NDI. Patients with NDI had lower birth weight, but there was no difference in gestational age at birth, severe intraventricular hemorrhage (IVH), necrotizing enterocolitis, or patent ductus arteriosus ligation compared with patients with no NDI. Ventilator days were greater in patients with NDI than in patients without NDI. More patients with NDI received furosemide and systemic corticosteroids and the hospital length of stay was longer than in patients with no NDI. Logistic regression modeling demonstrated that for every additional 100 g of birth weight the odds of NDI decreased by 35% and for every additional hospital day the odds of NDI increased by 1.3%. CONCLUSIONS: In our cohort of patients with moderate to severe BPD, the majority had no NDI, and low birth weight and length of hospital stay were associated with increased risk of developing NDI. This finding suggests that there are potentially modifiable factors associated with better neurodevelopmental outcomes in patients with BPD that deserve further study.

Detection of Cytomegalovirus in Intestinal Tissue of Infants with Necrotizing Enterocolitis or Spontaneous Intestinal Perforation.

OBJECTIVE: To determine the frequency of detection of cytomegalovirus (CMV) in surgical or autopsy intestinal tissue from infants with necrotizing enterocolitis (NEC) or spontaneous intestinal perforation (SIP) of the small bowel. STUDY DESIGN: This was a retrospective cohort study of infants in the neonatal intensive care unit at Nationwide Children's Hospital, Columbus, Ohio, with NEC (Bell stage ≥2B) or SIP from 2000 to 2016. Paraffin-embedded surgical or autopsy intestinal tissues were examined for CMV by polymerase chain reaction (PCR) and immunohistochemistry (IHC), and clinical characteristics of CMV-positive vs CMV-negative cases were compared. RESULTS: CMV was detected by PCR or IHC in 7 (4%) of 178 infants with surgical or autopsy- confirmed NEC (n = 6) or SIP (n = 1). Among 143 NEC cases (123 surgical, 20 autopsy), CMV was detected in 6 (4%): 4 (2 surgical, 2 autopsy) by both PCR and IHC, and 2 (surgical) by PCR only. Among 35 SIP cases (32 surgical, 3 autopsy), 1 (3%) surgical case was positive, by PCR only. CMV-associated NEC cases had lower median gestational age (24 vs 28 weeks; P = .02), birth weight (649 vs 1121 g; P = .04), and platelet count (16 000/mm3 vs 50 000/mm3; P = .018) compared with CMV-negative cases, respectively. No association was found with receipt of maternal milk, age at NEC diagnosis, male sex, cholestasis, or mortality. CONCLUSIONS: CMV was detected in intestinal tissue from 4% of NEC or SIP cases (NEC, 4%; SIP, 3%). Lower gestational age, lower birth weight, and thrombocytopenia were significantly associated with detection of CMV in NEC or SIP cases.

Regulation of Human Airway Epithelial Tissue Stem Cell Differentiation by ß-Catenin, P300, and CBP.

The wingless/integrase-1 (WNT)/ß-catenin signaling pathway is active in several chronic lung diseases including idiopathic pulmonary fibrosis, asthma, and chronic obstructive pulmonary disease. Although this WNT/ß-catenin pathway activity is associated with an increase in mucus cell frequency and a decrease in ciliated cell frequency, a cause and consequence relationship between signaling and cell frequency has not been established. We previously demonstrated that genetic stabilization of ß-catenin inhibited differentiation of mouse bronchiolar tissue stem cells (TSC). This study determined the effect of ß-catenin and its co-factors P300 (E1A-binding protein, 300 kDa) and cAMP response element binding (CREB)-binding protein (CBP) on human bronchial epithelial TSC differentiation to mucus and ciliated cells. We developed a modified air-liquid interface (ALI) culture system in which mucus and ciliated cell frequency is similar. These cultures were treated with the ß-catenin agonist CHIR99021 (CHIR) and antagonists to ß-catenin (XAV939), P300 (IQ1), and CBP (ICG001). We report that human TSC differentiation to mucus and ciliated cells can be divided into two stages, specification and commitment. CHIR treatment inhibited mucus and ciliated cell commitment while XAV939 treatment demonstrated that ß-catenin was necessary for mucus and ciliated cell specification. Additional studies demonstrate that a ß-catenin/P300 complex promotes mucus cell specification and that ß-catenin interacts with either P300 or CBP to inhibit ciliated cell commitment. These data indicate that activation of ß-catenin-dependent signaling in chronic lung disease leads to changes in mucus and ciliated cell frequency and that P300 and CBP tune the ß-catenin signal to favor mucus cell differentiation. Stem Cells 2018;36:1905-12.

Infant Pulmonary Function Testing and Phenotypes in Severe Bronchopulmonary Dysplasia.

BACKGROUND: The definition of severe bronchopulmonary dysplasia (sBPD) is based on respiratory support needs. The management of a patient with sBPD remains empirical and is highly variable among providers. Our objective in this study was to test the hypothesis that infant pulmonary function testing (iPFT) would reveal distinct phenotypes in patients with established sBPD during the initial NICU stay. METHODS: A prospective cohort study with data prospectively collected on infants with sBPD from May 1, 2003, to June 30, 2016. iPFT data were used to classify the patients as obstructive, restrictive, or mixed. RESULTS: The median gestational age at birth was 25 weeks (interquartile range [IQR], 24-27 weeks) and the median birth weight was 707 g (IQR, 581-925 g). At the time of iPFT, the median postmenstrual age was 52 weeks (IQR, 45-63 weeks), and the median weight was 4.4 kg (IQR, 3.7-6.0 kg). There were 56 (51%) patients with obstructive, 44 (40%) with mixed, and 10 (9%) with restrictive phenotypes. Moderate or severe obstruction was seen in 86% of the obstructive group and 78% of the mixed group. Of the restrictive patients, 70% had moderate and 30% had mild restriction. Bronchodilator response was seen in 74% of obstructive, 63% of mixed, and 25% of restrictive patients. CONCLUSIONS: Our findings reveal that sBPD as it is currently defined includes distinct phenotypes. Future researchers of diagnostic approaches to this population should consider the development of bedside tests to define phenotypes, and researchers in future therapeutic trials should consider the use of pulmonary function phenotyping in patient recruitment.

Pulmonary sequelae and functional limitations in children and adults with bronchopulmonary dysplasia.

Preterm infants with bronchopulmonary dysplasia (BPD) often suffer from life-long pulmonary impairments in pulmonary physical function. This review summarizes our current understanding of the chronic pulmonary impairments and physical functional limitations associated with BPD from preterm birth to adulthood. It also identifies opportunities for intervention in children and adults living with chronic lung disease (CLD) after preterm birth.

Dietary Docosahexaenoic Acid Improves Cognitive Function, Tissue Sparing, and Magnetic Resonance Imaging Indices of Edema and White Matter Injury in the Immature Rat after Traumatic Brain Injury.

Traumatic brain injury (TBI) is the leading cause of acquired neurologic disability in children. Specific therapies to treat acute TBI are lacking. Cognitive impairment from TBI may be blunted by decreasing inflammation and oxidative damage after injury. Docosahexaenoic acid (DHA) decreases cognitive impairment, oxidative stress, and white matter injury in adult rats after TBI. Effects of DHA on cognitive outcome, oxidative stress, and white matter injury in the developing rat after experimental TBI are unknown. We hypothesized that DHA would decrease early inflammatory markers and oxidative stress, and improve cognitive, imaging and histologic outcomes in rat pups after controlled cortical impact (CCI). CCI or sham surgery was delivered to 17 d old male rat pups exposed to DHA or standard diet for the duration of the experiments. DHA was introduced into the dam diet the day before CCI to allow timely DHA delivery to the pre-weanling pups. Inflammatory cytokines and nitrates/nitrites were measured in the injured brains at post-injury Day (PID) 1 and PID2. Morris water maze (MWM) testing was performed at PID41-PID47. T2-weighted and diffusion tensor imaging studies were obtained at PID12 and PID28. Tissue sparing was calculated histologically at PID3 and PID50. DHA did not adversely affect rat survival or weight gain. DHA acutely decreased oxidative stress and increased anti-inflammatory interleukin 10 in CCI brains. DHA improved MWM performance and lesion volume late after injury. At PID12, DHA decreased T2-imaging measures of cerebral edema and decreased radial diffusivity, an index of white matter injury. DHA improved short- and long-term neurologic outcomes after CCI in the rat pup. Given its favorable safety profile, DHA is a promising candidate therapy for pediatric TBI. Further studies are needed to explore neuroprotective mechanisms of DHA after developmental TBI.

Measuring End-Tidal Carbon Monoxide of Jaundiced Neonates in the Birth Hospital to Identify Those with Hemolysis.

BACKGROUND: End-tidal breath carbon monoxide (ETCOc) levels correlate with catabolism of heme, but until recently, this measurement was not readily available for application to neonatology practice. OBJECTIVES: We performed a prospective, multihospital, test-of-concept study where ETCOc was measured during the birth hospitalization of neonates with a total bilirubin (TB) value >75th percentile on the Bhutani bilirubin nomogram. This was done to test the feasibility and ease of use of this new device. METHODS: Neonates with an elevated ETCOc (with a >95th percentile reference interval previously established) were labeled as having 'hemolytic jaundice'. We recommended a follow-up TB check <24 h after hospital discharge to these families. RESULTS: One hundred and fifteen neonates were eligible for the study, the parents of 103 provided consent, and measurements were obtained for 100. Sixty-three had normal and 37 had elevated ETCOc values. By means of a direct antiglobulin test (DAT; Coombs), 11 of these 37 were found positive for ABO hemolytic disease; the remaining 26 had other etiologies. Thirty-six of the 37 with an elevated ETCOc had repeat TB monitoring <24 h after discharge home. None of the 100 were rehospitalized for jaundice treatment compared with a rate of 2.99 rehospitalizations per 100 control neonates who had a TB value >75th percentile (p = 0.079). CONCLUSION: ETCOc measurement is a feasible means of assessing hemolysis in jaundiced neonates during the birth hospitalization. When hemolysis is identified, parents are likely to comply with instructions to bring the infant for a TB checkup <24 h after discharge home.

Maternal tobacco smoke increased visceral adiposity and serum corticosterone levels in adult male rat offspring.

BACKGROUND: Maternal tobacco smoke (MTS) predisposes human and rat offspring to visceral obesity in early adulthood. Glucocorticoid excess also causes visceral obesity. We hypothesized that in utero MTS would increase visceral adiposity and alter the glucocorticoid pathway in young adult rats. METHODS: We developed a novel model of in utero MTS exposure in pregnant rats by exposing them to cigarette smoke from E11.5 to term. Neonatal rats were cross-fostered to control dams and weaned to standard rat chow through young adulthood (postnatal day 60). RESULTS: We demonstrated increased visceral adiposity (193%)*, increased visceral adipose 11-ß hydroxysteroid dehydrogenase 1 mRNA (204%)*, increased serum corticosterone (147%)*, and no change in glucocorticoid receptor protein in adult male MTS rat offspring. Female rats exposed to MTS in utero demonstrated no change in visceral or subcutaneous adiposity, decreased serum corticosterone (60%)*, and decreased adipose glucocorticoid receptor protein (66%)*. *P < 0.05. CONCLUSION: We conclude that in utero MTS exposure increased visceral adiposity and altered in the glucocorticoid pathway in a sex-specific manner. We speculate that in utero MTS exposure programs adipose dysfunction in adult male rat offspring via alteration in the glucocorticoid pathway.

Lung heparan sulfates modulate K(fc) during increased vascular pressure: evidence for glycocalyx-mediated mechanotransduction.

Lung endothelial cells respond to changes in vascular pressure through mechanotransduction pathways that alter barrier function via non-Starling mechanism(s). Components of the endothelial glycocalyx have been shown to participate in mechanotransduction in vitro and in systemic vessels, but the glycocalyx's role in mechanosensing and pulmonary barrier function has not been characterized. Mechanotransduction pathways may represent novel targets for therapeutic intervention during states of elevated pulmonary pressure such as acute heart failure, fluid overload, and mechanical ventilation. Our objective was to assess the effects of increasing vascular pressure on whole lung filtration coefficient (K(fc)) and characterize the role of endothelial heparan sulfates in mediating mechanotransduction and associated increases in K(fc). Isolated perfused rat lung preparation was used to measure K(fc) in response to changes in vascular pressure in combination with superimposed changes in airway pressure. The roles of heparan sulfates, nitric oxide, and reactive oxygen species were investigated. Increases in capillary pressure altered K(fc) in a nonlinear relationship, suggesting non-Starling mechanism(s). nitro-l-arginine methyl ester and heparanase III attenuated the effects of increased capillary pressure on K(fc), demonstrating active mechanotransduction leading to barrier dysfunction. The nitric oxide (NO) donor S-nitrosoglutathione exacerbated pressure-mediated increase in K(fc). Ventilation strategies altered lung NO concentration and the K(fc) response to increases in vascular pressure. This is the first study to demonstrate a role for the glycocalyx in whole lung mechanotransduction and has important implications in understanding the regulation of vascular permeability in the context of vascular pressure, fluid status, and ventilation strategies.

Hyperoxia increases hepatic arginase expression and ornithine production in mice.

Hyperoxic exposure affects the levels and activities of some hepatic proteins. We tested the hypothesis that hyperoxic exposure would result in greater hepatic .NO concentrations. C3H/HeN mice were exposed to >95% O(2) for 72 or 96 h and compared to room air-breathing controls. In contrast to our working hypothesis, exposure to >95% O(2) for 96 h decreased hepatic nitrite/nitrate NO(X) concentrations (10.9 +/- 2.2 nmol/g liver versus 19.3 +/- 2.4 nmol/g liver in room air, P < 0.05). The hepatic levels of endothelial NO synthase (eNOS) and inducible NOS (iNOS) proteins were not different among the groups. The arginases, which convert L-arginine to urea and L-ornithine, may affect hepatic NOS activities by decreasing L-arginine bioavailability. Hepatic ornithine concentrations were greater in hyperoxic animals than in controls (318 +/- 18 nmol/g liver in room air, and 539 +/- 64, and 475 +/- 40 at 72 and 96 h of hyperoxia, respectively, P < 0.01). Hepatic arginase I protein levels were greater in hyperoxic animals than in controls. Hepatic carbamoyl phosphate synthetase (CPS) protein levels and activities were not different among groups. These results indicate that increases in hepatic levels of arginase I in mice exposed to hyperoxia may diminish .NO production, as reflected by lower liver levels of NO(X). The resultant greater hepatic ornithine concentrations may represent a mechanism to facilitate tissue repair, by favoring the production of polyamines and/or proline.